The present invention relates to polynucleotides and polypeptides for increasing cuticular water permeability of a plant expressing same. More particularly the present invention relates to genetically modified plants capable of producing dehydrated fruits, such as tomato.
Aerial portions of higher plants are covered with a continuous extracellular layer of cuticle. The cuticle is a polymer matrix which is mostly composed of cutin monomers (primarily short-chain hydroxylated fatty acids) and various amounts of cuticular waxes (solvent-soluble lipids). Both the cutin and the wax components vary greatly in amount and composition between different plant species and plant organs (Holloway, 1982). Although the components and structure of plant cuticle as well as the biological and genetic regulation of its biosynthesis has been extensively investigated (Kolattukudy, 1980; Koornneef et al., 1989; Blee and Schuber, 1993; Arts et al., 1996; Negruk et al., 1996; Millar et al., 1997; Todd et al., 1999; Yaphremov et al., 1999; Flebig et al., 2000; Pruitt et al., 2000; Wellesen et al., 2001 Hooker et al., 2002; Chen et al., 2003; Kuns and Samuels, 2003; Kurata et al., 2003; Aharoni et al., 2004; Schnurr et al. 2004), the mechanisms controlling the differentiation and/or function of the cuticle remain to be characterized.
The tomato fruit cuticle is a thin layer with a 4-10 micron thickness with two unique structural properties (Wilson and Sterling, 1976). First, the cutin deposits are arranged in a laminar structure—the layers are assembled in parallel to the epidermis cells. The second property of the tomato fruit cuticle is that it does not contain any stomata, pores or channels. As a result, the water permeability of the tomato skin is very low and the fully ripe tomato fruit retains its water content. The water permeability of a number of other cuticles lacking stomata (astomatous) and the mechanism of water transport across them have been the subjects of numerous investigations (Schönherr, 1976a; Schönherr and Schmidt, 1979; Riederer and Schreiber, 2001). Apparently, both the cutin and wax components have an integrated effect against water loss from the organ. In some cases, the thickness of the cuticular layer is inversely proportional to diffusion of water across cuticular membranes (Lownds et al., 1993). However, frequently the cuticular wax component is primary in affecting plant water permeability. For example, removal of the epicuticular wax layer from tomato fruit cuticles by organic solvents increased their water permeability by a factor of 300 to 500, as evidenced by rapid plant dehydration (Schönherr, 1976b). Additional evidence for the role of cuticular waxes as a transpiration barrier in tomato fruits is the recently reported gene encoding the enzyme very-long-chain-fatty acid (VLFA) β-ketoacyl-CoA synthase (LeCER6, Vogg et al., 2004). This gene plays an important role in the synthesis of VFLA which are a major component in fruit cuticular wax. A loss of function mutation in this gene led to the reduction of n-alkanes and aldehydes with chain lengths beyond C30 in both leaf and fruit waxes. Tomato fruits with the LeCER6 mutation were characterized with a 4-fold increase in water permeability. Another factor affecting water permeability of tomato fruit cuticle is the presence of cracking on the cuticular surface. Fruit cracking has received much research attention (Cotner et al., 1969; Voisey et al., 1970; peet, 1992; peet and willits, 1995). Tomato fruits are affected by three main types of cracking: i) Concentric cracking (coarse cracking); ii) Radial cracking (splitting); and iii) Cuticle cracking (russeting) (Bakker, 1988). The first two types of cracking are deep and extended fissures that penetrate through the fruit pericarp and in addition to water loss also allow pathogen penetration and fruit decomposition.
Unlike radial or concentric cracks, cuticle cracks are superficial micro fissures of the cuticle that are generally confined to the cuticle and do not penetrate to the pericarp cells. The causes and circumstances leading to fruit cracking in tomatoes are mostly unclear and may be related to cuticular layer thickness (Emmons and Scott, 1998), shape of the underlying epidermis cells (Conter et al., 1969; Emmons and Scott, 1998), fruit shape (Considine and Brown, 1981), fruit size (Koske et al., 1980; Emmons and Scott, 1997), relative humidity around the fruit (Young, 1947; Tukey, 1959), strong foliage pruning (Ehret et al., 1993) and the tensile strength and extensibility of the epidermis (Bakker, 1988).
The occurrence of cracks in tomato fruit also has a significant genetic component, which is mainly expressed upon gene transfer from wild species of Lycopersicon. Fulton et al. (2000) described a trait, “Epidermal reticulation” (Er), and, using an advanced backcross QTL analysis strategy (with the wild type L. parviflorum) reported four QTLs affecting it. Cuticlar cracks also have been reported in Lycopersicon fruit derived from crosses of L. esculentum and other wild species such has L. hirsutum (WO 0113708) and L. penellii (Monforte et al., 2001).
Cracks in fruit cuticle, particularly extreme cracks which are visually evidenced as epidermal reticulation, due to the development of a suberized coating along the fissure (Monforte et al., 2001), are generally considered to be negative phenomenon due to the esthetic damages that lower fruit value (Tukey, 1959), as well as due to the loss of fruit moisture content. However, the economic potential of fruits that dehydrate while whole and while still attached to the vine, is high. Dehydrated tomato products comprise an important portion of the tomato industry. The production of tomato pastes, ketchup, and other processed tomato products is dependant on the energy-requiring steps of dehydration. In addition, “sun-dried” tomato fruit are prepared in a drying process which consists of dehydrating cut tomato fruit either in the sun or in drying ovens. Both sun-drying and oven drying may lead to losses in food quality. For example, levels of ascorbic acid, one of the major nutritional contributions of tomatoes in the human diet, decrease significantly in response to sun-drying or oven-drying (Ojimelukwe, 1994). Furthermore, the necessity to cut the tomato fruit in half before the drying process does not allow for the production of whole dried tomato fruit.
The present inventor has previously described dehydrated tomatoes having reduced water content using classical genetic breeding techniques (WO 01/13708). It is appreciated that the classical genetic breeding techniques are limiting to gene transfer within species or between closely related species of the same genus. Also, classical breeding is characterized by relatively large introgressions which include other undesirable genes closely linked to the gene of interest.
Introgressed cultivated tomato plants have been previously described by Eshed and Zamir (1985) having a genetic background (Introgression line IL4-4, i.e., resulting from an introgression extending from telomeric marker TG464 to centromeric marker CT50; ca20 cM) which may be associated with undesired traits. Similarly, Monforte et al. (2001) have described tomato plants having a similar genetic background derived from L. hirsutum [sub-near introgression lines TA1468, TA1469, TA1476 which span from, and including, TG464 to CT173 (approximately. 10 cM)] and which display numerous effects, including undesirable effects.
There is thus a widely recognized need for and it would be highly advantageous to have genetically modified plants with increased cuticular water permeability which are devoid of the above limitations.